Antioxidant and Anti-Inflammatory Effects of Herbal Formula SC-E3 in Lipopolysaccharide-Stimulated RAW 264.7 Macrophages

Abstract

SC-E3 is a novel herbal formula composed of five oriental medicinal herbs that are used to treat a wide range of inflammatory diseases in Korean traditional medicine. In this study, we sought to determine the effects of SC-E3 on free radical generation and inflammatory response in lipopolysaccharide- (LPS-) treated RAW 264.7 macrophages and the molecular mechanism involved. The ethanol extract of SC-E3 showed good free radical scavenging activity and inhibited LPS-induced reactive oxygen species generation. SC-E3 significantly inhibited the production of the LPS-induced inflammatory mediators, nitric oxide and prostaglandin E₂, by suppressing the expressions of inducible nitric oxide synthase and cyclooxygenase-2, respectively. SC-E3 also prevented the secretion of the proinflammatory cytokines, IL-1β, TNF-α, and IL-6, and inhibited LPS-induced NF-κB activation and the mitogen-activated protein kinase (MAPK) pathway. Furthermore, SC-E3 induced the expression of heme oxygenase-1 (HO-1) by promoting the nuclear translocation and transactivation of Nrf2. Taken together, these results suggest that SC-E3 has potent antioxidant and anti-inflammatory effects and that these effects are due to the inhibitions of NF-κB and MAPK and the induction of Nrf2-mediated HO-1 expression in macrophages. These findings provide scientific evidence supporting the potential use of SC-E3 for the treatment and prevention of various inflammatory diseases.

Figure 1

Effects of the three herbal formulas (SC-E1, SC-E2, and SC-E3) on the production of NO in LPS-stimulated RAW 264.7 macrophages. Cells were pretreated with various concentrations (50–300 μg/mL) of each formula for 1 h and then stimulated with LPS (1 μg/mL) for 18 h. NO production was determined using Griess reagent. (Significant versus LPS treatment, ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001.)

Figure 2

Effects of SC-E3 on ROS generation and on the productions of inflammatory mediators and cytokines in LPS-stimulated RAW 264.7 macrophages. (a) Effects of SC-E3 on viability. Cell viability was determined by MTT assay. Cells were treated with various concentrations of SC-E3 extract (50, 100, 300, or 500 μg/mL) for 24 h. Values were expressed as percentages of the nontreated control. (b) Effect of SC-E3 on ROS generation. Fold increases in intracellular ROS versus nontreated control were determined by measuring DCF fluorescence intensities. (c) Effects of SC-E3 on LPS-induced NO production. Cells were stimulated with 1 μg/mL of LPS, in the absence or presence of various concentrations (50, 100, 300, or 500 μg/mL) of SC-E3 for 18 h. Nitrite production was measured using Griess reagent. (d) Effects of SC-E3 on LPS-induced PGE₂ production. (Significant versus the nontreated control, ^###p < 0.001, significant versus LPS treatment, ^∗∗p < 0.01 and ^∗∗∗p < 0.001.)

Figure 3

Effects of SC-E3 on LPS-induced proinflammatory cytokine production. Extracellular levels of IL-1β (a), TNF-α (b), and IL-6 (c) were measured using ELISA kits. (Significant versus the nontreated control, ^###p < 0.001, significant versus LPS treatment, ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001.)

Figure 4

Effects of SC-E3 on the LPS-induced expressions of iNOS and COX-2 proteins in RAW 264.7 macrophages. Protein expressions of iNOS and COX-2 in RAW 264.7 macrophages incubated with different concentrations of SC-E3 (50, 100, 300, or 500 μg/mL) with or without LPS (1 μg/mL) for 24 h were assessed by western blot. The results showed that SC-E3 reduced the protein levels of iNOS (a) and COX-2 (b). (Significant versus nontreated control, ^###p < 0.001, versus LPS treatment, ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001.)

Figure 5

Effects of SC-E3 on MAPK pathway activation in LPS-stimulated RAW 264.7 macrophages. Cells were pretreated with different concentrations of SC-E3 (50, 100, 300, or 500 μg/mL) for 12 h and then stimulated with LPS (1 μg/mL) for 1 h. Western blotting was performed with antibodies for p-ERK, ERK, p-JNK, JNK, p-p38, and p38.

Figure 6

Effects of SC-E3 on NF-κB pathway activation by LPS. RAW 264.7 cells were pretreated with SC-E3 (300 μg/mL) for 18 h and then stimulated with LPS (1 μg/mL) for 1 h. (a) Cell lysates were subjected to western blot analysis to determine the protein levels of p-NF-κB and p-IκB-α. (b) The nuclear translocation of NF-κB was observed by immunofluorescence microscopy.

Figure 7

Effects of SC-E3 on the Nrf2/HO-1 signaling pathway in RAW 264.7 macrophages. (a) Induction of HO-1 by SC-E3. Cells were treated with different concentrations of SC-E3 (50, 100, 300, or 500 μg/mL) for 18 h. (b) Cells were treated with 300 μg/mL SC-E3 for the indicated times. (c) Nuclear accumulation of Nrf2 by SC-E3. Nrf2 was immunoblotted in the nuclear fractions of cells treated with 300 μg/mL of SC-E3 for the indicated times. (d) Immunofluorescence images of the nuclear translocation of Nrf2 induced by SC-E3. RAW 264.7 cells were treated with 300 μg/mL of SC-E3 for 3 h. (e) Blocking of the inhibitory effect of SC-E3 on LPS-induced NO production by SnPP (an HO-1 inhibitor). RAW 264.7 cells were pretreated with SC-E3 (300 μg/mL) for 1 h in the presence or absence of SnPP (50 nM, 30 min) and then stimulated with LPS (1 μg/mL) for 18 h. ^∗∗∗p < 0.001.

Figure 8

Identification of compounds from the ethanolic extract of SC-E3. (a) HPLC chromatogram of SC-E3 extract. The two compounds, geniposide and puerarin, were selected as marker compounds for quality control. The retention times of geniposide and puerarin were 12.183 and 11.830 min, respectively. (b) Representative GC-MS chromatogram for the analysis of fatty acids in SC-E3. Peaks: 1: puerarin, 2: geniposide, 3: pentadecanoic acid, 4: palmitic acid, 5: oleic acid, and 6: linoleic acid.